Opioids are essential to the management of acute and persistent pain, but their chronic use is associated with significant adverse effects, includin tolerance and dependence. There is now considerable information as to the physiological and molecular changes associated with chronic opioid use, but treatments that mitigate the problems are limited and generally ineffective. Unquestionably, the long lasting cellular changes that contribute to the development of tolerance and the manifestations of withdrawal are governed, at least partly, by stable changes in gene expression that are induced by pathways directly engaged during opioid receptor signaling. On the other hand, as manifestations of chronic opioid use, including psychological dependence, may persist long after the termination of opioid intake, we hypothesize that there are additional, maladaptive epigenetic mechanisms that retain transcriptional responses after the removal of the initial stimulus. Here, we propose t identify not only the transcriptional changes associated with tolerance and withdrawal, but also the epigenetic changes that we hypothesize induce and sustain them. We will use mouse models of morphine tolerance and withdrawal and we will focus our analysis on two brain regions, the locus ceruleus (LC) and the ventral tegmental area (VTA), both of which have been implicated in critical features of long term opioid use. We will perform genome wide, next generation sequence-based analysis of changes in the transcriptome (RNA-seq) and the epigenetic landscape (ChIP-seq) of FAC-sorted, pure neuronal populations. Importantly, we will extend our analysis to the 3-dimensional organization of the epigenome, which as is now recognized, provides an additional layer of epigenetic regulation that is likely more stable than chromatin and DNA post-translational modifications. To achieve the high-resolution, 3-dimensional and quantitative imaging of primary neurons isolated from the LC and VTA, we will use soft X-ray tomography (SXT). SXT, as we have recently demonstrated, is ideal for the study of nuclear architecture in primary neurons, and provides the most sensitive imaging method for generating an unbiased identification of the changes in chromatin organization and compaction associated with chronic opioid (morphine) use. This approach will be complemented by a genome wide chromatin conformation capture approach (Hi-C) and DNA FISH experiments. Thus, our proposed experiments will not only map the nuclear and epigenetic landscape of completely uncharted neuronal populations but will also have the potential to provide novel and fundamental insights into the epigenetic processes associated with tolerance and dependence. Together our findings will lay the foundation for the development of novel pharmacological interventions that may not only improve the management of pain by opioids, but also reduce the incidence of adverse side effects, including opioid abuse.